Summary/Abstract (Project 1) The central hypothesis of this project is that intrinsic structural and dynamic features of fVIII are responsible for its immunogenicity. The basis for this hypothesis is the remarkable immunogenicity of fVIII compared to other proteins. Additionally, during the early immune response to fVIII, fVIII immune complexes may amplify the immune response through Fc receptor or B cell receptor (BCR), antigen presentation or other mechanisms. To address this hypothesis, a better understanding of the structures and dynamics of fVIII and fVIII immune complexes is required. There are three Specific Aims in this project. Aim 1 is to develop X-ray crystallographic structures of fVIII and fVIII immune complexes. Development of atomic level resolution X-ray structures of fVIII, activated fVIII and fVIII immune complexes is fundamental to a better understanding of the immune response to fVIII. The available fVIII X-ray structures are not at atomic level resolution. Structures of immune complexes of the fVIII C2 domain are available. However, there are no structures of immune complexes with full-length or B domain-deleted fVIII. We have developed a high-expression platform that yields sub-gram quantities of a fVIII construct designated HP47 for crystallographic trials. In preliminary studies, we have crystals of HP47 fVIII that diffract beyond 3.2 resolution and are refining the structure. Aim 2 is to characterize fVIII antibody epitopes and fVIII immune complexes by hydrogen deuterium exchange mass spectrometry (HDX MS), electron microscopy (EM) and surface plasmon resonance spectroscopy. These methods are state-of-the-art complementary methods to characterize epitopes. HDX MS mapping studies of the C1 and C2 domain have been performed by our group. We have now obtained HDX coverage of the ~ 250-kDa full-length fVIII molecule, which will be used to map immunodominant epitopes throughout the fVIII molecule. Aim 3 is to characterize fVIII immune complexes by sedimentation velocity analytical ultracentrifugation (SV AUC). IgG immune complexes can amplify the immune response to the cognate antigen. However, the role of fVIII immune complexes in the immune response to fVIII has not been explored. A combination of two or more IgG molecules that recognize non-overlapping epitopes hypothetically can form large immune complexes with molecular weights extending to megadaltons. SV AUC is the method of choice for identifying and characterizing large protein-protein complexes. In preliminary studies, we have determined the stoichiometry of binary fVIII/anti-A2 MAb 4A4 and fVIII/anti-C2 MAb 3D12 complexes and the ternary fVIII/4A4/3D12 complex. These studies will be extended to characterize the stoichiometries of immune complexes formed by additional important classes of anti-fVIII MAbs. X-ray crystallographic, HDX MS, EM and SV AUC results be combined to develop models of fVIII IgG immune complexes. Additionally, the immune complexes characterized in Aim 3 will be tested in cellular and in vivo systems in collaboration with Project 2 for their possible contribution to the immunogenicity of fVIII.